Although guanidine and its derivatives have important biochemical and pharmacological activities, the molecular basis for the biological behavior of these compounds has not been clearly elucidated. On the basis of work done in this laboratory, hypotheses have been developed to describe two important aspects of quanidine derivative behavior: trans membrane transport and protein binding. Non ionic diffusion appears to be the major mechanism for membrane transport and may correlate with certain pharmacological activities such as hypoglycemia. Guanidine derivatives bind to the divalent metal cation binding site of at least one purified enzyme, pyruvate kinase, and display "calcium-like" specificity and activity. In the studies proposed in this application, the hypothesis of non-ionic diffusion across membranes will be explored in detail, using largely the mitochondrial system and measuring uptake both directly, with labelled guanidine derivatives, and indirectly, by effects on respiratory and ion-transporting functions. The n- alkylbiguanide series of analogues will be studied by these techniques, exploiting the effects of temperature, pH, long-chain free fatty acid and competing cations on biguanide uptake. The nature of the biguanide (and divalent metal ion) binding site in both pyruvate kinase and, ultimately, in mitochndria will be explored using a variety of probes, such as triethyltin, and modifiers of amino acid structure, such as dimethylpyrazole carbamidine, iodoacetate and photo oxidation. Binding to intact and modified protein will be measured directly using ultrafiltration techniques.